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J I Pedersen

Publications and source records attributed to J I Pedersen.

At least 91 records · Page 5Linked to original sources
PubMed1983

Formation of (4R)- and (4S)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A from Ochratoxin A by rabbit liver microsomes.

Three metabolites were formed from ochratoxin A in the presence of rabbit liver microsomal fractions and NADPH. They were isolated by extraction, thin-layer chromatography, and high-pressure liquid chromatography. Two of them were identified as (4R)- and (4S)-4-hydroxyochratoxin A. It is suggested on the basis of mass and nuclear magnetic resonance spectroscopy that the third metabolite is 10-hydroxyochratoxin A. The formation of the metabolites was inhibited by carbon monoxide and metyrapone and was stimulated when microsomes from phenobarbital-treated animals were used. The results suggest that cytochrome P-450 catalyzes the formation of these metabolites.

Animals↗
PubMed1983

Plasma clearance, transfer, and distribution of vitamin D3 from intestinal lymph.

The clearance of vitamin D3 from plasma after intravenous injection to rats of intestinal lymph labeled in vivo with radioactive vitamin D3 has been studied. The half-life of the injected vitamin D3 was about 6 min. In double-label experiments the half-life was the same for vitamin D3 as for vitamin A, which is removed from the circulation as chylomicron remnants. Fractionation of the blood plasma according to the density showed that 2 min after the injection of the lymph 30-95% of the radioactive vitamin D3 was recovered in plasma fractions with higher densities than chylomicron remnants (d greater than 1.019 g/ml). The percentage of vitamin D3 in the chylomicron fraction (d less than 1.006 g/ml) of the injected lymph was 66-95%. Taking this into account it could be calculated that the amount of radioactive vitamin D3 transferred from chylomicrons or chylomicron remnants to plasma with d greater than 1.019 g/ml within 2 min varied from 26 to 72%. The transferred radioactivity was recovered in a protein fraction with chromatographic mobilities identical to the binding protein for vitamin D and its metabolites. During the first 13 min after injection the amount of radioactivity recovered in the liver was less than 10%, but from 15 to 55 min the amount increased to about 50% of the injected dose. These findings suggest that the transfer of vitamin D3 from chylomicrons to the alpha-globulin fraction previously observed in vitro is of importance for the in vivo handling of vitamin D3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Absorption, distribution, and transport of vitamin D3 and 25-hydroxyvitamin D3 in the rat.

The absorption of [14C]vitamin D3 and 25-[3H]hydroxyvitamin D3 into the mesenteric lymph and portal vein after simultaneous instillation in rat duodenum has been studied. The appearance of both isotopes in the mesenteric lymph and portal vein during the first 2 h was negligible. After 2-4 h the isotopes appeared both in the lymph and portal vein. The appearance of 25-[3H]hydroxyvitamin D3 in the mesenteric lymph was faster than that of [14C]vitamin D3. Total recovery in the lymph during 24 h for [14C]vitamin D3 and 25-[3H]hydroxyvitamin D3 was 15.2 and 14.3%, respectively. While 80% of vitamin D3 in mesenteric lymph was recovered in the chylomicrons (d less than 1.006 g/ml), 94% of 25-hydroxyvitamin D3 was recovered in the protein fraction with d greater than 1.21 g/ml. Seventy-one percent of 25-hydroxyvitamin D3 in the protein fraction was associated with the alpha-globulins. Both vitamin D3 and 25-hydroxyvitamin D3 that were recovered in the chylomicrons (d less than 1.006 g/ml) were transferred in vitro to the lymph fraction with d greater than 1.006 g/ml. Less than 12% of 25-hydroxyvitamin D3 was recovered in the liver after intravenous injection of mesenteric lymph labeled with 25-[3H]hydroxyvitamin D3. Previously, under similar conditions 40% of administered vitamin D3 was recovered in the liver.

Animals↗
PubMed1983

Role of the 26-hydroxylase in the biosynthesis of bile acids in the normal state and in cerebrotendinous xanthomatosis. An in vivo study.

On the basis of different in vitro studies, we have previously suggested that the basic metabolic defect in the rare inherited disease cerebrotendinous xanthomatosis (CTX) is a lack of a hepatic mitochondrial C27-steroid 26-hydroxylase, involved in the normal biosynthesis of bile acids (1980. J. Clin. Invest. 65: 1418-1430; 1981. J. Lipid Res. 22: 191-200; 22: 632-640). In the present work, this hypothesis was tested in vivo. One patient with CTX and two control subjects received intravenously a mixture of [4-14C]7 alpha-hydroxy-4-cholesten-3-one and [6 beta-3H]7 alpha,26-dihydroxy-4-cholesten-3-one, steroids believed to be important precursors of chenodeoxycholic acid. The ratio between 14C and 3H in cholic acid and chenodeoxycholic acid isolated from bile of the CTX-patient was approximately 1/40 and 1/60 of those of the control subjects, respectively. Another patient with CTX and one control subject received a mixture of [4-14C]5 beta-cholestane-3 alpha,7 alpha-diol and [1,2-3H]5 beta-cholestane-3 alpha,7 alpha,26-triol, both possible precursors to chenodeoxycholic acid. In this case the 14C/3H ratio in cholic acid and chenodeoxycholic acid from the patient with CTX was 1/10 and 1/15, respectively, compared with that of the control subject. The most likely explanation for these findings is that very little of the 14C-precursors, i.e. without a 26-hydroxyl group, can be converted into cholic acid and chenodeoxycholic acid because of a defect of the 26-hydroxylase step. The results obtained are in accord with our previous findings in vitro. The results further underline the importance of the 26-hydroxylase pathway in the normal biosynthesis of cholic acid and chenodeoxycholic acid in man.

Adult↗
PubMed1983

Formation of cholic acid from 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid by rat liver peroxisomes.

In a previous study, it was shown that the peroxisomal fraction of rat liver, isolated by Percoll gradient centrifugation of a light mitochondrial fraction, was able to catalyze conversion of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid (Pedersen, J. I., and J. Gustafsson, 1980. FEBS Lett. 121: 345-348). In the present work, this peroxisomal THCA-oxidizing system has been studied in more detail. The peroxisomes were prepared by sucrose gradient centrifugation. By use of different marker enzymes, it was confirmed that the major part of the activity in the light mitochondrial fraction was located in the peroxisomes. The reaction was absolutely dependent on the presence of Mg2+, CoA, ATP, and NAD+ in the reaction medium. In addition to cholic acid, small amounts of 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid were detected as product. Provided the peroxisomes were preincubated with ATP and CoA, the reaction was linear with time up to 75 min. It was linear with peroxisomal protein and the pH optimum was 8. The reaction was stimulated by FAD (ca. 50%), by cytosolic protein (about twofold), by microsomal protein (about twofold), bovine serum albumin (about sevenfold), and by KCN (75% at 1 mM). In the absence of bovine serum albumin in the medium the K'm for the overall reaction was 1.4 X 10(-6) M and the maximum rate was 4.3 nmol X mg-1 X hr-1. In the presence of bovine serum albumin, the K'm increased to 6.3 X 10(-6) M and the maximum rate to about 32 nmol X mg-1 X hr-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

26-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol by isolated nonparenchymal cells and hepatocytes from rat liver.

The ability of isolated nonparenchymal and parenchymal rat liver cells to metabolize 5beta-cholestane-3alpha,7alpha,12alpha-triol and other bile acid intermediates has been investigated. Incubation of nonparenchymal cells with 5beta-cholestane-3alpha,7alpha,12alpha-triol resulted in the formation of one more polar product identified as 5beta-cholestane-3alpha,7alpha,12alpha,26-tetrol. The formation was linear with time up to 2 hr and with the number of cells, and showed saturation kinetics with respect to substrate concentration. The maximum rate of conversion was 90 pmol/10(6) cells per hr. Incubation of hepatocytes with the triol resulted in the formation of several more polar products. In addition to 5beta-cholestane-3alpha,7alpha,12alpha,26-tetrol, products with retention time on high pressure liquid chromatography identical with 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-26-oic acid and cholic acid were observed. The identity of these products was verified by combined gas-liquid chromatography-mass spectrometry. The rate of conversion was linear with time for about 10 min and saturation with respect to substrate concentration was not attained. At identical substrate concentrations, the total rate of conversion (12.5 nmol/10(6) cells per hr) was at least two orders of magnitude higher than with the nonparenchymal cells. Similar differences in rates of conversion were observed with other C(27)-bile acid intermediates. It is concluded that the nonparenchymal cells do not play any significant role in the conversion of bile acid intermediates, either under physiological conditions or under experimental conditions where such steroids have been administered intravenously.-Dueland, S., J. I. Pedersen, C. A. Drevon, and I. Björkhem. 26-Hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha-triol by isolated nonparenchymal cells and hepatocytes from rat liver.

Animals↗
PubMed1981

Uptake and 25-hydroxylation of vitamin D3 by isolated rat liver cells.

The physiological roles played by hepatocytes and nonparenchymal cells of rat liver in the metabolism of vitamin D3 have been investigated. Tritium-labeled vitamin D3 dissolved in ethanol was administered intravenously to two rats. Isolation of the liver cells 30 and 70 min after the injection showed that vitamin D3 had been taken up both by the hepatocytes and by the nonparenchymal liver cells. The relative proportion of vitamin D3 that accumulated in the nonparenchymal cells increased with time. Perfusion of the isolated rat liver with [3H] vitamin D3 added to the perfusate confirmed the ability of both cell types to efficiently take up vitamin D3 from the circulation. By a method based on high pressure liquid chromatography and isotope dilution-mass fragmentography it was found that isolated liver cells in suspension had a considerable capacity to take up vitamin D3 from the medium. About 2.5 fmol of vitamin D3 were found to be associated with each hepatocyte or nonparenchymal cell after 1 h of incubation. 25-Hydroxylation in vitro was found to be carried out only by the hepatocytes. The rate of hydroxylation was about the same whether the cells were isolated from normal or rachitic rats (3.5 and 4 pmol of 25-hydroxyvitamin D3 formed per h per 10(6) cells, respectively). The possibility that the nonparenchymal cells might serve as a storage site for vitamin D3 in the liver is discussed.

Animals↗
PubMed1981

Formation of (4R)- and (4S)-4-hydroxyochratoxin A from ochratoxin A by liver microsomes from various species.

Two metabolic products were formed from ochratoxin A by human, pig, and rat liver microsomal fractions in the presence of reduced nicotinamide adenine dinucleotide phosphate. They were isolated from the incubation mixture in the presence of pig liver microsomes by extraction, thin-layer chromatography, and high-pressure liquid chromatography Their structures are suggested to be (4R)- and (4S)-4-hydroxyochratoxin A on the basis of mass and nuclear magnetic resonance spectroscopy. Km and the maximum velocity for the formation of the two metabolites by human, pig, and rat microsomes were determined. Their formation was inhibited by carbon monoxide and metyrapone. The results indicate that the microsomal hydroxylation system is a cytochrome P-450 and that different species are involved in the formation of the two epimeric forms of 4-hydroxyochratoxin A.

Animals↗
PubMed1981

Side chain hydroxylation of C27-steroids and vitamin D3 by a cytochrome P-450 enzyme system isolated from human liver mitochondria.

The present study was undertaken to obtain information on the involvement of cytochrome P-450 in the 26-hydroxylation on bile acid intermediates and in the 25-hydroxylation of vitamin D3 in human liver mitochondria. Cytochrome P-450 was solubilized from human liver mitochondria and purified two times to a specific content of 0.125 nmol per mg protein. Furthermore, a ferredoxin was isolated from the mitochondria and partly purified. This iron-sulfur protein had properties similar to bovine adrenal ferredoxin. A mitochondrial NADPH-ferredoxin reductase was also isolated and purified to homogeneity. This enzyme was a flavoprotein with properties very similar to the bovine adrenal NADPH-ferredoxin reductase. The cytochrome P-450 preparation catalyzed 26-hydroxylation of C27-steroids and 25-hydroxylation of vitamin D3 when reconstructed with NADPH, the ferredoxin and the ferredoxin reductase. With different substrates the following turnover numbers (nmol product X nmol P-450(-1) X min-1) were found: cholesterol, 8; 5-cholestene-3 beta, 7 alpha-diol, 10; 7 alpha-hydroxy-4-cholesten-3-one, 23; 7 alpha, 12 alpha-dihydroxy-4-cholesten-3-one, 27; 5 beta-cholestane-3 alpha, 7 alpha-diol, 28; 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, 41; and vitamin D3, 0.16. The hydroxylation reactions were inhibited by CO and metyrapone. The human liver mitochondrial ferredoxin and ferredoxin reductase could be replaced by adrenal ferredoxin and adrenal ferredoxin reductase without reduction of activity, but they could not be replaced by microsomal NADPH-cytochrome P-450 reductase. It is concluded that human liver mitochondria contain cytochrome P-450 involved in the oxidation of the side chain of C27-steroids and vitamin D3.

Adult↗
PubMed1981

Cerebrotendinous xanthomatosis: defective liver mitochondrial hydroxylation of chenodeoxycholic acid precursors.

Oxidation of the side chain of 5 beta-cholestane-3 alpha, 7 alpha-diol, 7 alpha-hydroxy-4-cholesten-3-one, and 5-cholestene-3 beta, 7 alpha-diol has been studied in subcellular fractions of liver from a patient with cerebrotendinous xanthomatosis (CTX) and a control subject. All intermediates were efficiently 26-hydroxylated and further converted to the corresponding 26-carboxylated derivatives by the mitochondrial fraction of normal human liver. No such conversion was observed with the mitochondria from the liver of the CTX patient and the control subject. 12 alpha-Hydroxylation of the patient and the control subject. 12 alpha-Hydroxylation of the substrates was very efficient with the microsomal fractions from both subjects. Bases on these and previous findings (Oftebro, H., I. Björkhem, S. Skrede, A. Schreiner, and J. I. Pedersen. 1980. J. Clin. Invest. 65: 1481-1430), it i concluded that the metabolic defect in CTX is a complete lack of mitochondrial C27-steroid 26-hydroxylase. In CTX the precursors of chenodeoxycholic acid are first attacked by the microsomal 12 alpha-hydroxylase and subsequently by the microsomal 25-hydroxylase as an alternate route to cholic acid formation. This explains the increased ratio of cholic acid to chenodeoxycholic acid observed in the bile of these patients. In the normal liver the formation of both cholic acid and chenodeoxycholic acid involves a mitochondrial 26-hydroxylation.

Adult↗
PubMed1981

Assay of intermediates in bile acid biosynthesis using isotope dilution--mass spectrometry: hepatic levels in the normal state and in cerebrotendinous xanthomatosis.

The synthesis of 2H4-labeled 5 beta-cholestane-3 alpha, 7 alpha-diol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, 7 alpha-hydroxy-4-cholesten-3-one, and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one is described. A mixture of these compounds, together with 2H3-labeled 5-cholestene-3 beta, 7 alpha-diol, was added to extracts of different subcellular fractions of liver. After purification by high performance liquid chromatography and conversion into trimethylsilyl ethers, the amounts of different endogenous unlabeled steroids were determined by selected ion monitoring. In normal liver, the concentration of 5-cholestene-3 beta, 7 alpha-diol (about 0.1-0.2 microgram/ml protein) was higher than the concentration of the other steroids (about 0.01-0.05 microgram/mg protein). The concentration of the different steroids was highest in the microsomal fraction of the liver homogenate. In a liver sample from a patient with cerebrotendinous xanthomatosis (CTX), the amounts of the 12 alpha-hydroxylated steroids were considerably higher than in the normal liver. The levels of 7 alpha-hydroxy-4-cholesten-3-one and 5 beta-cholestane-3 alpha, 7 alpha-diol were similar or only slightly higher than in the liver of the control patients. The concentration of 5-cholestene-3 beta, 7 alpha-diol was very high in the mitochondrial fraction of the CTX-liver. The findings are in accordance with the previous demonstration that the basic metabolic defect in CTX is a lack of the mitochondrial 26-hydroxylase. The results are further compatible with the contention that 7 alpha,26-dihydroxy-4-cholesten-3-one is an important intermediate in the normal bile acid biosynthesis.

Bile Acids and Salts↗
PubMed1980

Formation of 4-hydroxyochratoxin A from ochratoxin A by rat liver microsomes.

Hydroxyochratoxin A was isolated and identified from the urine of rats after injection with ochratoxin A. By incubating ochratoxin A with rat liver microsomes and reduced nicotinamide adenine dinucleotide phosphate, one major (90%) and two minor metabolites, more polar than ochratoxin A, were formed. Thin-layer chromatography revealed that the major metabolite had Rf values identical to those of hydroxyochratoxin A in six different solvent systems. Formation of the metabolites in vitro was inhibited by carbon monoxide and by metyrapone, and the rate of formation increased after pretreatment of the rats with phenobarbital. A type I spectrum appeared upon binding of ochratoxin A to microsomes with a spectral dissociation constant (Ks) of 37.6 microM. These findings strongly suggest the involvement of a cytochrome P-450 in the hydroxylation of ochratoxin A by rat liver microsomes. Apparent Km and Vmax values for the formation of hydroxyochratoxin A were determined to 50 microM and 5.5 nmol/mg of protein per h, respectively.

Animals↗